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Abstract

We present a two-photon microscope that images the full extent of murine cortex with an objective-limited spatial resolution across an 8 mm by 10 mm field. The lateral resolution is approximately 1 µm and the maximum scan speed is 5 mm/ms. The scan pathway employs large diameter compound lenses to minimize aberrations and performs near theoretical limits. We demonstrate the special utility of the microscope by recording resting-state vasomotion across both hemispheres of the murine brain through a transcranial window and by imaging histological sections without the need to stitch.

Figures (8)

Fig. 1 Design issues of the ultra-large-field-of-view two-photon scanning microscope. (a) Illustration of scan-induced aberrations through a spherical singlet lens in a configuration that corresponds to a scan lens. Scanning across a lens maintains on-axis behavior in the direction perpendicular to the scan, yet results in off-axis behavior along the scan dimension, with a reduced effective focal length at increasing off-axis scan angles. (b,c) The on-axis scan beam (red rays) converges at the paraxial focal plane (plane iii, which would be conjugate to the sample plane in a scanning microscope) but the off-axis scan beam (green rays) converges closer to the lens with separate horizontal (plane i) and vertical foci (plane ii). The focal plane intensity maps explicitly show the resulting astigmatism. An intensity of exactly one is the diffraction limited peak value; the maximums for panel b are i: 0.016, ii: 0.035, and iii: 1.000 and those for panel c are i: 0.134, ii: 0.114, and iii: 0.014.

Fig. 2 Calculated performance of the ultra-large-field-of-view two-photon scanning system. (a) Unfolded schematic of the optical layout of the fully corrected system; element separations are drawn only approximately to scale and the detection system is shown in Fig. 3(a). (b) The predicted PSF with the beam on- and off-axis for the fully corrected system. The color scale of the calculated PSFs have a common normalization. (c) Plots of the axial and lateral extent of the PSF. (d) The theoretical focal volume, calculated as the ellipsoid enclosed by the predicted half-maximal two-photon intensity points along the optical axis and the half-maximal intensity ellipse in the focal plane, for the system with only the on-axis correction versus full system corrections for scan-induced aberrations. The limiting volume on axis is 10 µm3.

Fig. 3 Calculated performance of the detection system for the ultra-large-field-of-view scanning microscope. (a) Unfolded schematic of the optical layout of the full pathway; element separations are drawn only approximately to scale. (b) The predicted collection of rays at the plane of the active area of the photomultiplier tube for three exit angles from the back aperture of the objective.

Fig. 4 Beam characteristic of the ultra-large-field-of-view two-photon scanning microscope. (a) Experimental verification of beam collimation across the full range of scan angles utilizing an interferometric shear plate. The rotation of the interference fringes at large scan angles, found from the slopes of the fringes (yellow lines) for the center and x = 4.3 mm scan positions, is consistent with a residual beam divergence of less than 1 mrad. (b) The envelope of the interferometric autocorrelation of the laser pulses evaluated through the full optical path of the microscope at the focus of the objective. The calculated fit (red) to the envelope of the experimental data (blue) is consistent with an initial pulse width of 110 fs (FWHM) that is chirped to a final width of 400 fs.

Fig. 5 Performance of the ultra-large-field-of-view two-photon scanning microscope. (a) Two-photon intensity measurements from a uniform bath of fluorescein and accompanying map of contours of constant intensity. (b) Intensity profiles along the X- and Y-axes of the data in panel a. We further show intensity data obtained with the beam parked on-axis and the objective tilted about an axis in the plane of the back aperture; these measurements provide an upper bound on the intensity attainable with this objective. (c) Experimental measurements of the PSF as a function of scan angle. The color scale for the measured PSFs are all self-normalized. Although this represents a significant deviation from a flat field, the maximal residual beam convergence for the scan system remains relatively moderate, equivalent to a downstream focus of 20 m. (d, e) Plots of the axial and lateral extent of the PSFs. Red and blue data points represent the performance of the system under standard scanning conditions. Green data points are the PSF found by tilting the objective; these represent the objective-limited performance of the system. (f) The measured shift in the height of the focal spot as a function of scan position along with the calculated height based on a residual scan-dependent beam convergence.

Fig. 6 Two-photon imaging (λ0 = 800 nm) of vasomotion in cortical arterioles across both hemispheres of an awake, head-fixed mouse through dual transcranial windows. Blood plasma is stained with fluorescein dextran. (a) Maximally projected image stack across 500 µm of the preparation. The arbitrary-line-scan path (yellow) spans both hemispheres and operated at 71 Hz. The dark region in the middle corresponds to a physical mask placed over the remaining cranial bone above the midline. The expanded images are single planes in each hemisphere and serve to highlight the path of the line-scan through individual vessels whose diameters were concurrently monitored. (b) Vasomotor oscillations measured simultaneously from pial arteries in the right (green) and left (red) hemispheres. An expanded and overlaid view of the highlighted time band (gray) is shown on the right. (c) Spectral power of variation in diameter from the two arterioles and a venule (panel a) indicate vasomotion in the arterioles but not in the venule. Spectra were calculated from 540 s traces and a bandwidth (FWHM) of 0.03 Hz. The system noise (gray) was found by measuring the diameter of 8 to 20 µm fibers imbedded in clear cement and measured 3 to 4 mm off axis. The data represents an average over 24 measurements. (d) Cross-correlation of the diameter for the arterioles from panel b reveals strong synchrony of the arterial diameter oscillations across hemispheres. The time-lag at the peak is 0.0 ± 0.1 s. (e) The black curve is the magnitude of the spectral coherence between the two arterioles as calculated with a bandwidth of 0.04 Hz. The gray curve is the coherence for two fibers across an equivalent sized field.

Fig. 7 Volumetric two-photon imaging (λ0 = 800 nm) of the vasculature in one hemisphere of mouse cortex. Blood plasma is stained with fluorescein dextran. (a) Cortical vasculature through a thin skull window that is projected over 210 µm. (b) Descending planar images at different depths within the cyan box in panel a. Each image is the average of ten optical sections. The depth is limited by the laser power, 80 mW at the focus. (c) The expanded field within the cyan box in panel a shows a single planar field acquired at a depth of 110 µm below the pial surface and a scan path for functional imaging of blood flow. The segment along the vessel (magenta) tracks individual RBCs and that perpendicular to the vessel (green) reports the diameter. (d) Scan path imaging through the capillary. At mid-height in image, the broad segment with dark streaks indicate the passing of RBCs; the speed of the RBCs is inferred from the slop of the streaks. The thin segment in the lower portion of the image reports the diameter of the vessel.

Fig. 8 Two-photon imaging (λ0 = 900 nm) of a fixed and cleared coronal section from a mouse that expressed enhanced green fluorescent protein in a sparse subset of cortical and hippocampal neurons. The excitation wavelength was 900 nm. (a) Maximally projected image stack across 500 µm of a 1.0-mm thick slice of tissue; data obtained at 2.2 µm per Z-section. (b) Highlight of the hippocampal region; yellow box in panel a. (c) Highlight of the hippocampal cells from panel a; the Z-projections are across ~60 µm of tissue at different depths into the tissue section, as indicated, and the Y-projection is across ~15 µm of tissue with the corresponding axial bands demarcated.